The major objective of the research proposed herein is the purification and characterization of selectively deuterated thymidylate synthetase and dihydrofolate reductase. The enzymes will be isolated from a methotrexate-resistant strain of lactobacillus casei cultivated in either fully-deuterated media, or deuterated media which contains specific protonated amino acids. Proton magnetic resonance will be used as a tool to study the interactions of the deuterated enzyme analogs with protonated substrates, inhibitors, and activators. Covalent chromatography will be employed to separate the subunits of thymidylate synthetase for the purpose of comparing the catalytic and structural properties of the dimer and monomer. Subunit interactions which may occur upon the formation of the ternary complex between selectively deuterated thymidylate synthetase, 5-fluoro deoxyuridylate and methylenetetrahydrofolate will be investigated using nuclear magnetic resonance. The growth of deuterated L. casei may yield many other important deuterated biological molecules amenable to studies using nuclear magnetic resonance, neutron scattering and infrared spectroscopy. These techniques should be of general utility in the study of biomolecular structure, and with the development of recombinant DNA techniques, may even be extended to selectively deuterated mammalian proteins.